Cell surface receptors constitute a class of proteins which are responsible for receptor-mediated endocytosis of specific ligands. Basically, the receptors serve as escorts for ligand delivery to intracellular destinations.
Ligand delivery is generally achieved through coated regions on the plasma membrane called "coated pits." These pits continually invaginate and pinch off, forming "coated vesicles" in the cytoplasm. Coated pits and vesicles provide a pathway for receptor mediated endocytosis of specific ligands. The ligands that bind to specific cell surface receptors are internalized via coated pits, enabling cells to ingest large numbers of specific ligands without taking in correspondingly large volume of extracellular fluid. The internalized coated vesicles may or may not lose their coats and bind with other vesicles to form larger vesicles called "endosomes." In the endosome the ligand and the receptor are separated or "sorted." Endosomes which sort ligands and receptors are known as "compartment of uncoupling of receptor and ligand" or "CURL."
Endosomes may fuse with primary lysosomes, where their contents are digested, or they may be delivered to other intracellular destinations. The receptor proteins are generally not digested, but are rather recycled to the cell membrane surface through a process called "exocytosis," or transferred to early or late endosomes via multivesicular bodies. The entire pathway is referred to as the "receptor trafficking pathway."
Some receptors deliver their ligand directly to the cytoplasm or other specific intracellular locations. Perhaps one of the most studied receptor trafficking pathways is that of iron transport. In this pathway, a serum carrier protein, transferrin, binds iron and transports it to transferrin receptors on the plasma membrane surface. After binding and internalization, via coated pits, the resulting vesicle combines first with early endosomes and then with late endosomes. This process results in the gradual drop in pH in the vesicle. The drop in pH causes the transferrin carrier protein to lose its affinity to iron. When this occurs, the iron translocates through the membrane of the vesicle and joins the intracellular pool of enzymes. The transferrin receptor may then recycle to the cell surface where it may repeat the process.
Other receptors may deliver their ligand directly to the lysosomes for digestion. For example, the epidermal growth factor ("EGF") receptor delivers its ligand directly to a lysosome for degradation (Prog. Histochem. Cytochem. 26:39-48,1992). The EGF receptor may recycle to the cell surface depending on its state of phosphorylation (Cancer Treat. Rep. 61:139-160, 1992; J. Cell. Biol. 116:321-330, 1992).
A single receptor may utilize more than one receptor trafficking pathway within the same cell. For example in polarized cells, such as specialized transport epithelia cells, membrane trafficking is distinct between apical and basal sides of the cell (Sem. Cell. Biol. 2:387-396, 1991). Moreover, non-polarized epithelia cells may simultaneously follow two separate sorting pathways.
The control or regulation of cell surface receptors may be achieved by a variety of techniques. Regulation of cell surface receptors may be accomplished, at a very basic level, by the binding of naturally occurring ligands. As discussed above, receptor binding of a ligand will generally trigger the internalization of the ligand-receptor complex. Such internalization may desensitize the cell to further ligand binding. (J. Immunol. 150:3161-9, 1993; Mol. Endocrinol. 6:2090-102, 1992; J. Cell. Physiol. 154:281-8, 1993; Receptor 1:13-32, 1990-91; Biochem. J. 288:55-61, 1992; J. Immunol. 148:2709-11, 1992; J. Cell. Physiol. 148:24-34, 1991). This type of regulation, however, is transient in nature and does not result in diminution of biologic response.
Regulation of cell surface receptors may also be accomplished by administration of receptor antagonists or agonists. Receptor antagonists are organic protein or peptide ligands generally derived through empirical structure-function studies, or through the use of detailed knowledge of ligand and receptor interaction. Essentially, an antagonist may constitute any molecule with similar binding activity to a natural ligand, but incapable of producing the biological response normally induced by the natural ligand. Thus, the antagonist competitively blocks receptor activity. With a competitive antagonist, the regulation of receptor activity is dependent upon both the antagonist's affinity for the receptor, as well as its extracellular concentration over time. Receptor agonists are protein or peptide ligands derived in a similar manner as antagonists. Essentially, an agonist may constitute any molecule which binds to the receptor in a manner superior to that of the natural ligand.
One receptor of particular interest is the vitamin B.sub.12 receptor. As has been demonstrated in experimental in vitro data, pre-clinical animal models, and patient studies, vitamin B.sub.12 is a co-enzyme necessary in cell division, as well as cellular metabolism, in proliferating normal and neoplastic cells. Insufficient vitamin B.sub.12 causes cellular division to be held in abeyance and ultimately may result in apoptosis. The nutrient is generally derived from dietary intake and is transported throughout the body complexed to transport proteins. The complex of transport protein and vitamin B.sub.12 is recognized by a cellular receptor which internalizes the complex and releases the vitamin intracellularly. The overall process has been reviewed in GUT 31:59, 1991. Vitamin B.sub.12 is taken in through the diet. Binding proteins in the saliva (R-binder) and gut (intrinsic factor-(IF)) complex vitamin B.sub.12 after release from endogenous binding proteins by action of enzymes and low pH in the stomach. Vitamin B.sub.12 is transferred across the intestinal epithelium in a receptor specific fashion to transcobalamin II (TcII). The vitamin B.sub.12 /transcobalamin II complex is then transported throughout the body and recognized by receptors present on dividing cells, internalized and released within the cell where it is utilized by certain enzymes as a co-factor.
The high affinity receptor in dividing tissues or cells responsible for internalization of vitamin B.sub.12 recognizes transcobalamin II complexed with vitamin B.sub.12. The vitamin B.sub.12 /TcII receptor recognizes only the vitamin B.sub.12 /TcII complex and not the serum transport protein or the vitamin alone. The receptor is undetectable on non-dividing cells; the mechanism for supplying non-dividing cells with vitamin B.sub.12 is poorly understood. However, it is known that more vitamin B.sub.12 is required during cell division than during metabolism, and that the vitamin B.sub.12 /TcII receptor is the only high affinity means for cellular uptake of vitamin B.sub.12 during cell division. When stimulated to divide, cells demonstrate transient expression of this receptor leading to vitamin B.sub.12 uptake which precedes actual DNA synthesis (J. Lab. Clin. Med. 103:70, 1984). Vitamin B.sub.12 receptor levels may be measured by binding of .sup.57 Co-vitamin B.sub.12 complexed to transcobalamin II (present in serum) on replicate cultures grown in chemically defined medium without serum. No receptor mediated uptake occurs in the absence of carrier protein.
Dividing cells, induced to differentiate, lose receptor expression and no longer take up vitamin B.sub.12. More importantly, leukemic cells, deprived of vitamin B.sub.12, will stop dividing and die (Acta Haemat. 81:61, 1989). In a typical experiment, leukemic cell cultures were deprived of serum for 3 days, and then supplemented either with serum (a source of vitamin B.sub.12) or a non-metabolizable analogue of vitamin B.sub.12 and cultured up to five days. Cell cultures supplemented with vitamin B.sub.12 continued to grow, whereas those deprived of the active nutrient stopped growing and die.
Based on these observations, it has been suggested that whole body deprivation of vitamin B.sub.12 may be useful in the treatment of cancer or other disorders characterized by uncontrolled growth of cells. Moreover, because of the critical role played by vitamin B.sub.12 -containing enzymes in cell division, it is believed that vitamin B.sub.12 deprivation may be used in combination with chemotherapeutic drugs which inhibit cellular replication. For example, when vitamin B.sub.12 depletion was combined with methotrexate, the two modalities together were more efficient in depleting folate levels in leukemic cells than either alone (FASEB J. 4:1450, 1990; Arch. Biochem. Biophvs. 270:729, 1989; Leukemia Research 15:165, 1991). Folates are precursors in the production of DNA and proteins. In typical experiments, cultures of leukemic cells were exposed to nitrous oxide for several hours to convert the active form of endogenous vitamin B.sub.12 to an inactive form. Replicate cultures were then left without further treatment, or additionally treated with methotrexate. Cellular folate levels were measured three days later. Cells treated with the combination (i.e., both methotrexate and inactive vitamin B.sub.12) showed a more striking decrease in cellular folate levels than with either of the two approaches alone. This combination also results in a higher cell kill in vitro. When this approach was applied to the treatment of highly aggressive leukemia/lymphoma in animal models (Am. J. Haematol. 34:128,1990; Anticancer Res. 6:737, 1986; Cancer Chemother. Pharmacol. 17:114, 1986; Br. J. Cancer 50:793, 1984), additive or synergy of anti-tumor action was observed, resulting in prolonged remissions and cures.
A key finding in the experiments described above was that short-term (hours to days), whole body depletion of vitamin B.sub.12 can act synergistically with chemotherapeutic drugs (such as methotrexate and 5-FU) to inhibit tumor growth and treat animals with leukemia/lymphoma. Despite synergistic anti-tumor activity, there was no toxicity attributable to the short-term vitamin B.sub.12 depletion for proliferating normal cells. This combination therapy was demonstrated in multiple animal models. Observations in patients have indicated that long-term (months to years) vitamin B.sub.12 depletion is required to produce significant normal tissue toxicity. Even in those cases, subsequent infusion of vitamin B.sub.12 can readily reverse symptomology (Br. J. Cancer 5:810, 1989).
Because of the promise of this therapeutic approach, various methods have been sought to efficiently and controllably perform a temporary depletion of vitamin B.sub.12. Such methods, however, affect all of the body's stores of vitamin B.sub.12. They include dietary restriction, high doses of vitamin B.sub.12 analogues (non-metabolizable-competitive antagonists which act as enzyme inhibitors), and nitrous oxide (transformation of vitamin B.sub.12 to inactivate form). These different methods have been used in culture systems and in animals to deplete vitamin B.sub.12. The most efficient and the most utilized method has been the inhalation of nitrous oxide (laughing gas). Animals are maintained typically under an atmosphere of 50% to 70% of nitrous oxide for periods from a few hours to a few days, causing the conversion of endogenous vitamin B.sub.12 into an inactive form. This methodology has been utilized in combination with drugs for therapy of leukemia/lymphoma. A further method for vitamin B.sub.12 depletion involves infusion of a non-metabolizable analogue of vitamin B.sub.12 which essentially dilutes out the active form. This form of therapy is not specific for dividing cells but affects liver dependent metabolic processes. Another approach includes restricting the dietary intake of vitamin B.sub.12. This method, however, requires very long periods of dietary restriction and is offset by hepatic storage of vitamin B.sub.12. All of these methods suffer from problems of specificity, since they affect both vitamin B.sub.12 -dependent growth as well as basal metabolism, and therefore are not particularly suited to the development of anti-proliferative pharmaceutical products.
In view of the biological importance of cell surface receptors, receptor-controlling agents have emerged as a class of pharmaceutical drugs. Moreover, with the advent of genetic engineering for the isolation and amplification of genes for cell surface receptors, as well as computer programs to model the interactions between ligands and receptors (i.e., "rational" drug design), the production of receptor-controlling drugs has been significantly enhanced.
To date, many months or even years of scientific research, as well as significant financial resources, are required to produce new receptor antagonists or agonists. To speed up this process, new screening technologies have been developed which utilize peptide or antibody recombinant libraries (see, e.g., Gene 73:305, 1988; Proc. Nat. Acad. Sci. (USA) 87:6378, 1990; Biochromatography 5:22, 1990; Protein Engineering 3:641, 1989). While library screening does not require the same degree of knowledge of a specific receptor/ligand system, it does involve an intensive screening effort utilizing functional receptor-specific assays. Moreover, the initial compounds identified by such screening programs are generally only precursors to the development of therapeutic products through more typical structure-functional assessments.
While antagonists and agonists are generally capable of regulating a biological response, the surface receptors which bind such ligands are continually being re-expressed on the cell surface. Thus, effective regulation by antagonists or agonists must rely on a relatively high and sustained serum concentration in order to bind the new surface receptors continually being expressed on the cell surface.
Accordingly, there is a need in the art for agents which bind cell surface receptors and thus regulate biological responses associated therewith, and which further effect normal cellular trafficking of the bound receptor. There is also a need in the art for agents which, when bound by a cell surface receptor and internalized, promote retention of the receptor within the cell. Moreover, there exists a need for methods relating to the administration of such agents to regulate a biological response. The present invention fulfills these needs and provides further related advantages.